Defects in the cytoskeletal architecture of the heart are associated with dilated cardiomyopathies. Here, we focus on cytoskeletal elements that stabilize the transverse (t) tubules of cardiac muscle and that link them to the nearby sarcoplasmic/endoplasmic reticulum (SR/ER). Recent results suggest that the interface between the t-tubules and the SR/ER is composed of at least two different protein arrays. One is composed of the classic mediators of excitation-contraction coupling, voltage-gated Ca channels in the t-tubule and Ca release channels (ryanodine receptors) in the SR/ER. The second is composed of other integral membrane proteins of the t-tubule and the SR/ER, linked by a membrane-bound cytoskeletal network of spectrin and ankyrin. Spectrin and its associated proteins are also likely to bind to and stabilize the t-tubular membrane at sites that are not closely apposed to the SR/ER membrane. We propose to test the hypothesis that the cytoskeleton of t-tubules is composed of two distinct spectrin networks, one that helps organize and stabilize the t-tubular membranes, and another that links them to the SR/ER in a complex that is regulated by phosphorylation. We will use a combination of molecular, cell biological, ultrastructural, proteomics and physiological approaches to address this hypothesis. We have five specific aims: (1) to characterize alphaII-cardi+, an alternatively spliced form of spectrin that is selectively expressed in the heart, and the complexes it forms; (2) to characterize other spectrins and the complexes they form at the cardiac t-tubule membrane; (3) to identify the spectrins and ankyrins that link the t-tubule to the SR/ER membrane, and the proteins with which they interact; (4) to study the effects of phosphorylation on the spectrin complexes at t-tubules; (5) to assess the effects of altering the spectrin network at t-tubules on the morphology and physiology of cardiocytes. Each of these aims is supported by our preliminary results, which suggest that t-tubules and their interactions with the SR/ER are coordinated by unique spectrin complexes, regulated by local signaling cascades. Defining these interactions and the mechanisms that control them should provide unique insights into how the heart functions normally, and how changes in cytoskeletal architecture at the t-tubules can result in cardiomyopathy.